This invention relates to a novel ganglioside ceramidase and a process for producing it. More particularly, it relates to a ganglioside ceramidase having physico-chemical properties shown at least by the following substrate specificity and enzyme action.
Substrate specificity; It has substrate specificity at least for GD1a, GM1, GM2 and GM3.
Enzyme action; It acts at least on GD1a, GM1, GM2 and GM3 to catalyzes the reaction of hydrolysis of ganglioside to lysoganglioside and a fatty acid.
Gangliosides are found in cell surface membranes of mammalian tissues and organs and are especially rich in the neural tissues. Gangliosides are sphingolipids produced by the linking of ceramides to oligosaccharides containing at least one acidic saccharide residue, for example, N-acetylneuraminic acid residue. As these gangliosides, for example, GD1a, GM1, GM2 and GM3 have been reported.
This GM2 ganglioside is represented by the structure: GalNAc 1-4(NeuAc 2-3)Gal 1-4Glc 1-1' cer (wherein Gal means galactose, GalNAc means N-acetylgalactosamine, Glc means glucose, NeuAc means N-acetylneuraminic acid and cer means residue of ceramide). GM2 ganglioside accumulates in the lysosomal storage disease, so-called Tay-Sachs disease and is degraded in the lysosome by a series of enzymes, beginning at the non-reducing terminal of the molecule. However, it has been reported that a specific activator is required for catabolism of GM2 ganglioside with .beta.-hexosaminidase A. [Proc. Natl. Acad. Sci. USA. 75, 3979-3983 (1978), Hoppe-Seyler's Z. Physio. Chem. 360, 1837-1849 (1979), J. Biol. Chem. 256, 6234-6240 (1981)]. GM2 ganglioside is not decomposed with exo-.beta.-N-acetyl hexosaminidase isolated from molds such as Aspergillus niger [Hoppe-Seyler's Z. Physio. Chem. 364, 821-829 (1983)], Penicillium oxalicum [Agric. Biol. Chem. 49, 611-619 (1985)] and Mucor fragilis [Applied Environ. Microbiol. 51, 1019-1023 (1986)].
Furthermore, an enzyme which hydrolyzes linkage between sphingosine base and fatty acid of ceramide is called ceramidase; EC 3.5.1.23 [J. Biol. Chem. 241, 3731-3737 (1966), Biochemistry 8, 1692-1698 (1969), Biochim. Biophys. Acta 176, 339-347 (1969), Science 178, 1100-1102 (1972)], but this ceramidase does not hydrolyze the linkage between a sphingosine base and the fatty acid of a glycolipid like ganglioside.
As explained above, exo-N-acetyl hexosaminidase isolated from molds, does not decompose GM2 ganglioside and an enzyme which decomposes GM2 ganglioside has not been known.
The inventors have succeeded in the isolation and purification of an enzyme which produces lysoganglioside GM2 and a fatty acid by hydrolysis of GM2 ganglioside as a substrate from culture of microorganism N285 strain belonging to genus Nocardia collected and isolated from forest soil in Himeji-shi in Hyogo-ken, Japan. This enzyme has been decided to be a novel enzyme having physico-chemical properties and has a novel enzyme action in that it does not utilize Gal-cer, Glc-cer Lac-cer, Gb3-cer and asialoGM1 as substrate, but utilizes at least GD1a, GM1, GM2 and GM3 as substrate and hydrolyzes ceramide in the molecule of this ganglioside into sphingosine base and fatty acid to produce lysoganglioside and fatty acid. This enzyme has been named ganglioside ceramidase.
Features of culture of the above N285 strain belonging to genus Nocardia in the various culture media according to visual observation and microscope observation are as follows:
A. Microscopical Observation
Short hyphae of 5 -10 .mu.m formed by static culture in a meat extract medium, a peptone medium or a glucose-peptone-yeast extract medium. Simply branched. Straight or curved, but short rod form and readily aggregated in case of liquid shaking culture.
B. State of Growth on the Media
(1) Sucruose.multidot.Nitrate Agar Medium
Growth good.
White peach color colonies formed in the form of convex circle or circle. Hyphae short.
(2) Glucose.multidot.Asparagine Agar Medium
Growth somewhat good.
White brown colonies formed in amorphous with periphery edge.
(3) Starch Agar Medium
Growth somewhat good.
White brown colonies formed in large circle.
(4) Tyrosine Agar Medium
Growth somewhat good.
White brown colonies formed in amorphous, glanules with convex circle.
Hyphae long.
(5) Yeast.multidot.Malt Agar Medium
Growth somewhat good.
Milky white colonies formed in somewhat large convex circle.
In these media (1)-(5), no production of pigment at the back of hyphae.
C. Physiological Properties
Aerobic, non-motile.
Growing temperature range: 15.degree.-30.degree. C.
Liquefaction of gelatin: Negative.
Hydrolysis of starch: Negative.
Production of melanine-like pigment
(tyrosine agar medium): Negative.
Nitrate reduction ability: Positive.
Denitrification reaction: Negative.
MR test, VP test, production of indole:
Negative.
Utilization of inorganic nitrogen source:
Possible (Growth is possible in all media other than those of succinic acid or glutamic acid).
Production of acid from sugar: Negative with glucose, galactose, mannose and sucrose.
Catalase: Positive.
Assimilation of carbon source: (on Pridham and Gottlieb agar medium)
(1) L-arabinose: - PA1 (2) D-xylose: - PA1 (3) D-glucose: + PA1 (4) D-fructose: + PA1 (5) Sucrose: + PA1 (6) L-rhamnose: - PA1 (7) Raffinose: - PA1 (8) Mannitol: -
This strain N285 was identified as a strain belonging to genus Nocardia from the feature such as form of colonies and simple branch of hyphae and was named Nocardia sp. N285. The strain has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, bearing FERM P-9540 and transferred to deposition under the Budapest Treaty as FERM BP-1830.